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Prenatal LHRH Neurons in Nasal Explant Cultures Express Estrogen Receptor ß Transcript

Cellular and Developmental Neurobiology Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892

Address all correspondence and requests for reprints to: Susan Wray, Chief, Cellular and Developmental Neurobiology Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Building 36, Room 5A-21, Bethesda, Maryland 20892-4156. E-mail: . swray{at}codon.nih.gov

	Abstract

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Sex steroids influence LHRH neuronal activity, exerting a negative or positive feedback action, depending on the reproductive state of the animal. Recent evidence indicates that LHRH neurons possess the estrogen receptor ß (ERß) subtype postnatally, suggesting that estrogen may act, in part, directly on LHRH neurons. In this study, we identified ERß transcript in prenatal LHRH neurons as a function of age. Single-cell cDNA pools were made from LHRH neurons maintained for 7, 14, and 28 d in vitro (div). Screening of the cDNA pools by PCR with ERß-specific primers revealed ERß-positive LHRH neurons at all three ages. However, the number of LHRH cells coexpressing ERß transcript decreased dramatically between 14 (6/10) and 28 div (1/10). None of the LHRH cells were positive for ER transcript. These results indicate that developing LHRH neurons express the transcript for ERß and suggest that continued expression of ERß is either a characteristic of LHRH neurons that may require cues from the central nervous system and/or periphery or predetermined to be maintained in a subpopulation of LHRH neurons.

	Introduction

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THE LHRH NEURONAL system is essential for initiation and maintenance of reproductive function in vertebrates (reviewed in Refs. 1 and 2). Pulsatile release of LHRH into the hypophyseal portal system affects the synthesis and secretion of gonadotropins and consequently activation of gonadal function (3). The mechanisms that precisely regulate LHRH secretion are poorly defined, but estrogen is certainly one of the most critical factors affecting LHRH neuronal activity (4). Estrogen can exert either a negative or positive feedback action on LHRH neuronal activity, depending on the reproductive state of the animal. Despite the clear relation between estrogen levels and LHRH release, the mode of action of estrogen on LHRH neuronal activity is still unclear. Numerous studies examining estrogen receptor (ER) expression in LHRH neurons led to the general hypothesis that estrogen altered LHRH neuronal activity trans-synaptically or via glial interactions (4). Although the presence of ER in LHRH neurons is still controversial (4, 5), recent evidence indicates that adult LHRH neurons possess the ERß subtype (6, 7, 8, 9). These new findings suggest that estrogen may act, in part, directly on LHRH neurons.

Tissue cell culture systems of LHRH neurons derived from prenatal nasal regions (10, 11, 12, 13, 14, 15, 16, 17) provide an alternative model for studies designed to evaluate the inter- and intracellular mechanisms regulating LHRH neuronal activity. Similar to the developing embryo in vivo, cell cultures containing LHRH neurons derived from prenatal nasal regions turn on LHRH gene expression, peptide synthesis, and processing (11, 18). Recent work indicates that when maintained for long periods of time in vitro, LHRH cells in nasal cultures exhibit pulsatile-like secretion (15, 16, 17). Thus, in nasal explants, prenatal LHRH cells continue to differentiate with respect to secretory profiles. Importantly, in mouse nasal explants, large numbers of LHRH neurons are maintained in a concentrated area and can be identified in situ because of their migrational behavior (12, 19, 20, 21). As early as 3 d in vitro (div), LHRH neurons within these explants have migrated in a unidirectional path away from the tissue mass onto the substratum (11, 19). Therefore, molecular studies on cDNAs obtained from individual LHRH neurons can be performed (19, 20, 21). In the present study, we examined primary LHRH neurons at different in vitro ages for the expression of ERß transcript.

The experiments reported in this paper demonstrate that primary LHRH neurons, devoid of central nervous system (CNS) influences, express ERß transcripts as early as 7 div and that this profile changes by 28 div, with the number of LHRH cells expressing ERß transcript decreasing. These data indicate that LHRH cells, outside the CNS, turn on ERß expression and suggest that continued expression of this transcript is either maintained only in a subpopulation of LHRH neurons or characteristic of LHRH neurons that require cues from the CNS and/or periphery.

	Materials and Methods

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In vitro cell preparations
Nasal regions were cultured as previously described (11). Briefly, embryos were obtained from timed pregnant animals in accordance with NIH guidelines. Nasal pits of embryonic d 11.5 (E11.5) staged NIH-swiss mice were isolated under aseptic conditions and refrigerated for 1 h in Gey’s balanced salt solution (Life Technologies, Inc., Grand Island, NY) enriched with glucose (Sigma, St. Louis, MO). Nasal explants were adhered onto coverslips by a plasma (Cocalico Biologicals, Inc., Reamstown, PA)/thrombin (Sigma) clot. The explants were maintained in defined serum-free medium (SFM) (22) at 37 C with 5% CO₂. On culture d 3, fresh media containing uridine (5 mg/ml) + 5'-fluoro-2-deoxyuridine (2 mg/ml) (8 x 10^-5 M, Sigma) was given to inhibit proliferation of dividing olfactory neurons and nonneuronal explant tissue. On culture d 6 and every 2 d afterward, the media was changed to fresh SFM. Explants were used for experiments on culture d 7, 14, and 28. In addition, two immortalized LHRH cells lines were examined, NLTs (23) and GT1-7s (24). The immortalized cell lines were grown in DMEM (Life Technologies, Inc.) with 20% fetal calf serum. Cells were placed in SFM (22) for 24 h before use.

Cell isolation and generation of cDNA libraries for examination of ERs
Nasal explants were washed twice with 1x PBS (without Mg⁺, Ca²⁺) and placed in 2 ml of the same solution. Explants were observed under an inverted microscope (Nikon, Melville, NY), and LHRH-like neurons were identified by their bipolar morphology, association with outgrowing axons, and location within the explant. At each time point (7, 14, and 28 div), single LHRH-like cells (n = 10/group) were isolated from at least two different cultures using a micromanipulator fitted with a pulled microcapillary (FHC, Bowdinham, ME). The LHRH cell lines were trypsinized [0.5 ml trypsin and 10 ml Hanks’ calcium-free media (BioWhittaker, Inc., Walkersville, MD)] and the contents of the flask spun down (10 min, 2000 rpm). The media was removed and RNA extraction was performed using Totally RNA kit (Ambion, Inc., Austin, TX) according to directions. Briefly, lysis buffer was added to each flask followed by an equal volume of phenol:chloroform:IAA (25:24:1). The mixture was centrifuged (13,000 rpm, 10 min). The aqueous layer was placed in a fresh tube and precipitated with sodium acetate (3 M) and 100% cold ethanol. The pellet was washed with cold ethanol and left overnight at -20 C. The next day the pellet was washed again with cold ethanol and then resuspended in 100 µl nuclease-free water.

Complementary DNA libraries were generated as previously described (19). Briefly, 1 µg total brain RNA (Ambion, Inc.), ovary RNA (Ambion, Inc.), or cell line RNA, or a single cell was placed into a reaction mixture containing 4 µl cold lysis buffer [for 100 µl mix: 20 µl of Moloney murine leukemia virus buffer 5x (Life Tech/Invitrogen, Carlsbad, CA), 76 µl H₂O, 0.5 µl Nonidet P-40 (Amersham Pharmacia Biotech, Piscataway, NJ), 1 µl PrimeRNase inhibitor (5'3' Inc., Boulder, CO), 1 µl RNAguard (Amersham Pharmacia Biotech), 2 µl freshly made 1/24 dilution of stock primer mix (10 µl each 100 mM deoxynucleotide triphosphate (dNTP) (Life Tech/Invitrogen), 10 µl 50 OD/ml pd (T) 19–24, 30 µl H₂O)]. The mixture was incubated (65 C, 1 min; ice, 1 min; room temperature, 2 min). A 1:1 (volume, 0.5 µl) mix of avian myeloblastosis virus and Moloney murine leukemia virus-reverse transcriptases (Life Tech/Invitrogen) was added (37 C, 15 min; 65 C, 10 min). Then 5 µl stock tailing buffer [0.5 µl terminal deoxynucleotidyl transferase (Roche, Indianapolis, IN) and 4.5 µl tailing mix; 100 µl 5x (Life Tech/Invitrogen) terminal deoxynucleotidyl transferase buffer, 3.75 µl 100 mM deoxy-ATP, 146.25 µl H₂O] was added (37 C, 15 min; 65 C, 10 min). Samples were kept on ice until PCR.

PCR mix [10 µl of 10x PCR buffer II (Applied Biosystems, Foster City, CA), 10 µl of 25 mM MgCl₂ (Applied Biosystems), 0.5 µl of 20 mg/ml BSA (Roche), 1 µl each 100 mM deoxynucleotide triphosphate (Life Tech/Invitrogen), 1 µl 5% Triton (Sigma), 5 µg AL1 primer (ATTGGATCCAGGCCGCTCTGGAC-AAAATATGAATTC[T]24), 2 µl Ampitaq (Applied Biosystems), and 57.5 µl H₂O] was prepared on ice. Then 90 µl were added to each RT/tailing reaction containing 10 µl template. PCR was performed for 25 cycles in a DNA thermal cycler (Applied Biosystems; 94 C for 1 min, 42 C for 2 min, 72 C for 6 min with 10-sec extension time at each cycle). After the first 25 cycles, 1 µl AmpliTaq was added and 25 more cycles of PCR were performed under the same conditions minus the 10-sec extensions. The resulting product was phenol-chloroform extracted, then ethanol precipitated, and an aliquot run on a 1.5% agarose gel.

PCR analysis
Based on the technique used to generate the cDNA pools, 3' untranslated region (UTR) biased primers are necessary for PCR analysis. Primers were designed with the 5'-primer being less than 500 bases from the polyA site and the 3' primer close to, but not into, the polyA region. All designed primers were screened using BLAST to ensure specificity of binding. Three different primer sets were used for ERß to ensure that negative results were not due to technical parameters such as poor primer binding. Primers used were: LHRH (5'-ACTGGTCCTATGGGTTGCGCCCTG-3', 5'-CGGGGCCAGTGGACAGTACATTCG-3'), ßIII-tubulin (5'-GAGGACAGAGCCAAG-TGGAC-3', 5'-CAGGGCCAAGACAAGCAG-3'), ER (5'-TTTCTGTCCAGCACCTTG-AA-3', 5'-CTCAGATCGTGTTGGGGAAG-3'), and ERß (5'-CCATCTCTTTGCCCACT-TGG-3', 5'-GGTCGATTTGATGACCACACC-3'; 5'-GGCCTGTGAGGTAGGAATGCG-3'). For each reaction, 24.8 µl nuclease-free H₂O, 4.0 µl 10x PCR buffer (Applied Biosystems), 3.2 µl of 25 mM MgCl₂ (Applied Biosystems), 2.5 µl dNTP mix (10 µl of each dNTP, 360 µl H₂O), 0.5 µl Amplitaq Gold (Applied Biosystems) were mixed. Primer (2 µl) was added to the mixture (final concentration 100–500 nmol) and 1 µl template cDNA. Samples were run on RoboCycler (Stratagene, La Jolla, CA): 10 min at 94 C prerun; 30 sec (1 min) 94 C; 30 sec (1 min) 55 C or 65 C, depending on the primers; 2 min 72 C for 40 cycles; and 10 min 72 C postrun. Amplified products were run on a 1.5% agarose gel. Specific bands were observed in all control total brain and ovary lanes, and no bands were seen in water lanes. Each set of primers was run three times/cDNA pool (immortalized cells and single cells). PCR results positive in at least two of three runs were scored as expressing the appropriate transcript.

Immunocytochemistry
Nasal explants were immunocytochemically stained as previously described (11). Briefly, explants were fixed with 4% formaldehyde for 1 h, washed with PBS, incubated for 1 h in 10% normal goat serum/0.3% Triton X-100, washed several times in PBS, and then incubated in LHRH antibody (1:2500, SW1) overnight at 4 C. The next day, cultures were washed several times in PBS, incubated in biotinylated secondary antibody (1:500 in PBS/0.3% Triton X-100; Vector Laboratories, Inc., Burlingame, CA) for 1 h, washed with PBS several times, and processed for avidin-biotin-horseradish peroxidase/3'3-diaminobenzidine histochemistry (11).

	Results

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Bipolar cells in the periphery of the nasal explants, associated with outgrowing fibers, were removed (Fig. 1). RT-PCR was performed following a protocol known to generate a 3' UTR biased proportional cDNA pool (19). The sensitivity of this procedure has been shown to be reliable at 10 copies of mRNA/cell (21). As predicted from their morphology and location (11), the majority of the cells removed from the explants were found to be LHRH-positive neurons. Only cDNA pools positive for both ß-tubulin transcript and LHRH transcript were used in further experiments. The cDNA pools from single LHRH cells were then examined for the presence of ER and ERß transcripts (Fig. 2). At 7 and 14 div, 4 of 10 and 6 of 10 LHRH cells expressed ERß transcript, respectively. By 28 div, only 1 of 10 LHRH cells was ERß positive. All 30 LHRH cells, independent of age, were negative for ER transcript. The cDNAs from both immortalized cell lines were positive for ERß transcript and negative for ER transcript (Fig. 2). Brain and ovary cDNAs were positive for both ERß and ER transcripts (Fig. 2).

View larger version (152K): [in this window] [in a new window]   Figure 1. Large numbers of LHRH cells are present in nasal explants and can be identified in situ. A, Photomicrograph of a nasal explant maintained for 7 div and then immunocytochemically stained for LHRH. Numerous LHRH-positive neurons (arrows) can be seen in the periphery of explant. The solid line delineates the main nasal tissue from the periphery of the explant. The main nasal tissue consists of the olfactory pit (OP), midline nasal cartilage (MNC), and intervening mesenchymal areas. Bipolar LHRH-like cells in the periphery of the explant are identified in situ (B, arrow), and removed from the explant (C, arrow) with a microcapillary pipette (D). Note: the cell in B that has been removed in C is not the same cell shown in D. Bar, 40 µm in A.

View larger version (120K): [in this window] [in a new window]   Figure 2. LHRH neurons express ERß subtypes. Representative gel documentation of PCR products from single-cell RT-PCR performed on individual cells extracted from the periphery of nasal explants. Top row, cDNAs produced from polyA targeted, AL1 primer, PCR amplification; remaining rows show products produced by PCR amplification of above cDNAs using primers specific for LHRH; ß-tubulin; ER subtype; and ERß subtype. Brain (B, first column) and ovary RNA (O, second column) were used as starting material for positive controls. No specific bands were detected in water (W, third column). The cDNAs from LHRH cells maintained for 7, 14, and 28 div are shown in columns 4–6. Two immortalized LHRH cell lines (NLTs and GT1-7) were also examined (column 7). ERß transcript, but not ER transcript, was detected in primary LHRH cells at all three ages.

	Discussion

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The nasal explants used in these experiments were generated from E11.5 mouse embryos, a stage during which LHRH neurons are postmitotic, expressing LHRH mRNA and protein, and beginning to migrate out of the nasal pit (25, 26). The sex of the animal at E11.5 from which the explants were generated was not known. However, ERß-positive and -negative LHRH cells were found in a single explant, indicating that the results obtained were not dependent on the sex of the animal. The primers used to detect the ERß transcript reside within the last exon of the ERß transcript, within the 657-bp 3' UTR. To date, this region is present in all splice variants of ERß that bind estrogen (27, 28); thus, negative LHRH cells were most likely not the result of alternative splicing. We have previously shown that this method of cDNA generation and PCR analysis is reliable at 10 copies of mRNA/cell (21). Therefore, prenatal LHRH cells maintained in vitro in SFM are a heterogeneous population with respect to expression of ERß transcript.

LHRH neurons were examined for ERß and ER transcript at 7, 14, and 28 div. In addition to prenatal LHRH cells, two immortalized LHRH cell lines, the NLTs and GT1-7s, were also examined. All of the primary LHRH neurons, as well as the two LHRH cell lines, were negative for ER transcript. The presence of ER-like protein in LHRH neurons within the brain of postnatal mice has been reported (5). Certainly the discrepancy in these results could be due to the age of the LHRH cells and/or the location of the LHRH cells examined (brain vs. nose; in vivo vs. in vitro). The presence of ER transcript and protein has recently been reported for GT1-7 cells (29, 30). In the experiments reported in this paper, the immortalized cell lines were transferred to a defined SFM, identical with that used for the nasal explants (12). In contrast, ER protein was identified in GT1-7s grown in charcoal-stripped serum medium (29, 30), which, although low in steroids, still contains many other serum factors. Thus, the presence or absence of ER in GT1-7 cells appears to be dramatically influenced by serum factors or the removal thereof.

We have shown that by 7 div, LHRH neurons in nasal explants are postmigratory and possess many characteristics of well-differentiated neurons (11, 12, 18). Chronologically cells at 7, 14, and 28 div would be approximately equivalent to LHRH cells from animals of ages E18.5, postnatal d 7 and 21, respectively. At 7 and 14 div, 40–60% of the LHRH cells expressed ERß transcripts. At approximately E18 in rats, sexual differentiation occurs with a surge of testosterone aromatized to estrogen in the male (31). The expression of ERß by many LHRH neurons during prenatal development may be important for sexual differentiation to take place. By 28 div, only 10% of the LHRH cells were found to contain ERß mRNA. In vivo, 11–17% of LHRH neurons in prepubertal/adult female mice were found to express ERß transcripts (6). ERß protein in LHRH neurons in mice has not yet been reported, either prenatally or postnatally. However, a much higher percent of LHRH neurons in ovariectomized and ovariectomized/estradiol-treated rats expressed ERß transcripts (67–74%) (8) and ERß protein (52–84%, 8, 9). Whether there is a physiological difference between mouse and rat or a technical factor that accounts for the observed differences found between ERß expression in LHRH cells in these two species remains to be determined. In this study we found that the percent of LHRH cells that expressed the ERß transcript decreased as a function of age. Because LHRH cells from all three ages were run in parallel, it is unlikely that a technical factor explains the lower value found in these experiments at 28 div. With this in mind, it is striking that the percent of LHRH cells expressing ERß at 28 div is similar to that found for prepubertal/adult mice in vivo (6). However, before generalized statements can be made about species differences, more work on the role of ERß subtypes on LHRH function in each species is clearly needed.

In summary, this investigation has shown that prenatal LHRH neurons in embryonic nasal explants express ERß transcript and that expression of this transcript in LHRH neurons decreases over time. The expression of ERß by many LHRH neurons during prenatal development may be important for sexual differentiation to occur. In addition, the decrease in ERß expression in LHRH neurons observed over time may indicate that either a subpopulation of LHRH neurons are predetermined to maintain expression of ERß or that continued expression of ERß is a characteristic of the LHRH neuron that requires cues from the CNS and/or periphery.

	Footnotes

 
Present address for N.S.: The Johns Hopkins University, Department of Human Genetics, Baltimore, Maryland 21205.

Abbreviations: CNS, Central nervous system; div, days in vitro; dNTP, deoxynucleotide triphosphate; E11.5, embryonic d 11.5; ER, estrogen receptor; SFM, serum-free medium; UTR, untranslated region.

Received January 8, 2002.

Accepted for publication March 19, 2002.

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